Vesicular glutamate transporter type 3 (VGLUT3) containing neuronal elements were characterized using antibodies to VGLUT3 and molecular cell markers. All VGLUT3-positive somata were immunoreactive for CCK, and very rarely, also for calbindin; none was positive for parvalbumin, calretinin, VIP or somatostatin. In the CA1 area, 26.8 ± 0.7% of CCK-positive interneuron somata were VGLUT3-positive, a nonoverlapping 22.8 ± 1.9% were calbindin-positive, 10.7 ± 2.5% VIP-positive and the rest were only CCK-positive. The patterns of coexpression were similar in the CA3 area, the dentate gyrus and the isocortex. Immunoreactivity for VGLUT3 was undetectable in pyramidal and dentate granule cells. Boutons colabelled for VGLUT3, CCK and GAD were most abundant in the cellular layers of the hippocampus and in layers II-III of the isocortex. Large VGLUT3-labelled boutons at the border of strata radiatum and lacunosum-moleculare in the CA1 area were negative for GAD, but were labelled for vesicular monoamine transporter type 2, plasmalemmal serotonin transporter or serotonin. No colocalization was found in terminals between VGLUT3 and parvalbumin, vesicular acetylcholine transporter and group III (mGluR7a,b; mGluR8a,b) metabotropic glutamate receptors. In stratum radiatum and the isocortex, VGLUT3-positive but GAD-negative boutons heavily innervated the soma and proximal dendrites of some VGLUT3- or calbindin-positive interneurons. The results suggest that boutons coexpressing VGLUT3, CCK and GAD originate from CCK-positive basket cells, which are VIP-immunonegative. Other VGLUT3-positive boutons immunopositive for serotonergic markers but negative for GAD probably originate from the median raphe nucleus and innervate select interneurons. The presumed amino acid substrate of VGLUT3 may act on presynaptic kainate or group II metabotropic glutamate receptors.